T.A. Burns

Insulin resistance selectively alters cell-surface glucose transporters but not their total protein expression in equine skeletal muscle.

BACKGROUND Insulin resistance (IR) has been widely recognized in humans, and more recently in horses, but its underlying mechanisms are still not well understood. The translocation of glucose transporter 4 (GLUT4) to the cell surface is the limiting step for glucose uptake in insulin-sensitive tissues. Although the downstream signaling pathways regulating GLUT translocation are not well defined, AS160 recently has emerged as a potential key component. In addition, the role of GLUT12, one of the most recently identified insulin-sensitive GLUTs, during IR is unknown. HYPOTHESIS/OBJECTIVES We hypothesized that cell-surface GLUT will be decreased in muscle by an AS160-dependent pathway in horses with IR. ANIMALS Insulin-sensitive (IS) or IR mares (n = 5/group). METHODS Muscle biopsies were performed in mares classified as IS or IR based on results of an insulin-modified frequently sampled IV glucose tolerance test. By an exofacial bis-mannose photolabeled method, we specifically quantified active cell-surface GLUT4 and GLUT12 transporters. Total GLUT4 and GLUT12 and AS160 protein expression were measured by Western blots. RESULTS IR decreased basal cell-surface GLUT4 expression (P= .027), but not GLUT12, by an AS160-independent pathway, without affecting total GLUT4 and GLUT12 content. Cell-surface GLUT4 was not further enhanced by insulin stimulation in either group. CONCLUSIONS AND CLINICAL IMPORTANCE IR induced defects in the skeletal muscle glucose transport pathway by decreasing active cell-surface GLUT4.

Evaluation of Four Diagnostic Tests for Insulin Dysregulation in Adult Light‐Breed Horses

Background Several tests have been evaluated in horses for quantifying insulin dysregulation to support a diagnosis of equine metabolic syndrome. Comparing the performance of these tests in the same horses will provide clarification of their accuracy in the diagnosis of equine insulin dysregulation. Objectives The aim of this study was to evaluate the agreement between basal serum insulin concentrations (BIC), the oral sugar test (OST), the combined glucose‐insulin test (CGIT), and the frequently sampled insulin‐modified intravenous glucose tolerance test (FSIGTT). Animals Twelve healthy, light‐breed horses. Methods Randomized, prospective study. Each of the above tests was performed on 12 horses. Results Minimal model analysis of the FSIGTT was considered the reference standard and classified 7 horses as insulin resistant (IR) and 5 as insulin sensitive (IS). In contrast, BIC and OST assessment using conventional cut‐off values classified all horses as IS. Kappa coefficients, measuring agreement among BIC, OST, CGIT, and FSIGTT were poor to fair. Sensitivity of the CGIT (positive phase duration of the glucose curve >45 minutes) was 85.7% and specificity was 40%, whereas CGIT ([insulin]45 >100 μIU/mL) sensitivity and specificity were 28.5% and 100%, respectively. Area under the glucose curve (AUC g0‐120) was significantly correlated among the OST, CGIT, and FSIGTT, but Bland–Altman method and Lins concordance coefficient showed a lack of agreement. Conclusions Current criteria for diagnosis of insulin resistance using BIC and the OST are highly specific but lack sensitivity. The CGIT displayed better sensitivity and specificity, but modifications may be necessary to improve agreement with minimal model analysis.

Lamellar events related to insulin-like growth factor-1 receptor signalling in two models relevant to endocrinopathic laminitis

BACKGROUND Insulin dysregulation, obesity, and exposure to high-nonstructural carbohydrate (NSC) forage are risk factors for equine metabolic syndrome-associated laminitis (EMSAL); high systemic insulin concentrations in EMSAL are proposed to induce cellular dysregulation in the digital lamellae through activation of the insulin-like growth factor-1 receptor. OBJECTIVES To use a dietary challenge model (DCM) and a euglycaemic-hyperinsulinaemic clamp (EHC) model to assess lamellar growth factor-related signalling. STUDY DESIGN Lamellar phospho (P)-protein concentrations of signalling proteins important in growth factor-related signalling were assessed in 2 models: 1) lean and obese ponies on a low- or high-NSC diet; and 2) EHC model using Standardbred horses. METHODS Ponies stratified for body condition (lean [LN, n = 11] and obese [OB, n = 11]) were exposed to a low-NSC diet (LO, n = 5 per group for LN LO and OB LO) or a high NSC diet (HI, n = 6 per group for LN HI and OB HI groups) for 7 days. For the EHC model, horses were administered insulin (constant rate infusion [6 mIU/kg bwt/min] combined with 50% dextrose, EHC group, n = 8)] or saline (0.57 mL/kg bwt/h, CON group, n = 8) for 48 h. Immunoblotting was employed to assess concentrations of activated/phosphorylated and total protein for members of the PI3K/Akt/mTORC1 and Ras/ERK pathways in lamellar samples from both models. RESULTS In the DCM, lamellar P-(Ser 240/244) RPS6 was increased in OB HI ponies (vs. OB LO, P<0.05); positive correlations existed (P<0.05; r>0.5) between Day 7 basal serum insulin concentrations and lamellar concentrations of P-p70S6K and P-(Ser 240/244) RPS6. In the EHC model, lamellar concentrations of P-Akt, P-p70S6K, P-ERK 1/2, P-p90RSK, and both P-(Ser 235/236) and P-(Ser 240/244) RPS6 were increased in the EHC group (vs. CON, P<0.05). MAIN LIMITATIONS The primary limitations of this study are the small number of animals per group in the DCM study, and the fact that many animals did not develop laminitis as that was not the endpoint of either study. CONCLUSIONS These results support further investigation of mTORC1/RPS6 signalling as a potential therapeutic target(s) in EMSAL. The Summary is available in Chinese - see Supporting Information.

Steroids, steroid precursors, and neuroactive steroids in critically ill equine neonates

Hypothalamic-pituitary-adrenal axis (HPAA) dysfunction has been associated with sepsis and mortality in foals. Most studies have focused on cortisol, while other steroids have not been investigated. The objectives of this study were to characterise the adrenal steroid and steroid precursor response to disease and to determine their association with the HPAA response to illness, disease severity, and mortality in hospitalised foals. All foals (n=326) were classified by two scoring systems into three categories: based on the sepsis score (septic, sick non-septic [SNS] and healthy) and the foal survival score (Group 1: 3-18%; Group 2: 38-62%; Group 3: 82-97% likelihood of survival). Blood concentrations of adrenocorticotropic hormone (ACTH) and steroids were determined by immunoassays. ACTH-cortisol imbalance (ACI) was defined as a high ACTH/cortisol ratio. Septic foals had higher ACTH, cortisol, progesterone, 17α-OH-progesterone, pregnenolone, and androstenedione concentrations as well as higher ACTH/cortisol, ACTH/progesterone, ACTH/aldosterone, and ACTH/DHEAS ratios than SNS and healthy foals (P<0.01). Foals with DHEAS of 0.4-5.4ng/mL were more likely to have ACI (OR=2.5). Foals in Group 1 had higher ACTH, aldosterone, progesterone, and cortisol concentrations as well as ACTH/cortisol, ACTH/progesterone, and ACTH/DHEAS ratios than foals in Groups 2 and 3 (P<0.01). High progesterone concentrations were associated with non-survival and the cutoff value below which survival could be predicted was 23.5ng/mL, with 75% sensitivity and 72% specificity. In addition to cortisol, the response to the stress of illness in foals is characterised by the release of multiple adrenal steroids. DHEAS and progesterone were good predictors of HPAA dysfunction and outcome in hospitalised foals.

Effects of Common Equine Endocrine Diseases on Reproduction

Endocrine diseases, such as equine metabolic syndrome and pituitary pars intermedia dysfunction, are common in domesticated horse populations, and the frequency with which these diseases are encountered and managed by equine veterinary practitioners is expected to increase as the population ages. As clinicians learn more about the effects of these diseases on equine reproductive physiology and efficiency (including effects on reproductive seasonality, ovulation efficiency, implantation, early pregnancy loss, duration of pregnancy, and lactation), strategies and guidelines for improving fertility in affected animals continue to evolve. It is hoped that further research will establish these recommendations more firmly.

A novel model to assess lamellar signaling relevant to preferential weight bearing in the horse

Supporting limb laminitis (SLL) is a devastating sequela to severe unilateral lameness in equine patients. The manifestation of SLL, which usually only affects one limb, is unpredictable and the etiology is unknown. A novel, non-painful preferential weight bearing model designed to mimic the effects of severe unilateral forelimb lameness was developed to assess lamellar signaling events in the supporting limb (SL). A custom v-shaped insert was attached to the shoe of one forelimb to prevent normal weight bearing and redistribute weight onto the SL. Testing of the insert using a custom scale platform built into the floor of stocks confirmed increased distribution of weight on the SL compared with the unloaded forelimb (UL) and the contralateral (CH) and ipsilateral (IH) hind limbs in six Standardbred horses. In a second part of the study, eight healthy Standardbred horses were fitted with the insert and tied with consistent monitoring and free access to hay and water for 48 h, after which the lamellae were harvested. Real-time qPCR was performed to assess lamellar mRNA concentrations of inflammatory genes and immunoblotting and immunofluorescence were performed to assess lamellar protein concentration and cellular localization of hypoxia-related proteins, respectively. Lamellar mRNA concentrations of inflammatory signaling proteins did not differ between SL and either CH or IH samples. HIF-1α concentrations were greater (P < 0.05) in the SL compared to the CH. This work establishes an experimental model to study preferential weight bearing and initial results suggest that lamellar hypoxia may occur in the SL.

Effect of Intravenous Administration of Cobalt Chloride to Horses on Clinical and Hemodynamic Variables

Background Cobalt chloride (CoCl2) is administered to racehorses to enhance performance. The purpose of this study was to evaluate the clinical, cardiovascular, and endocrine effects of parenterally administered CoCl2. Objectives To describe the effects of weekly intravenous doses of CoCl2 on Standardbred horses. Animals Five, healthy Standardbred mares. Methods Prospective, randomized, experimental dose‐escalation pilot. Five Standardbred mares were assigned to receive 1 of 5 doses of CoCl2 (4, 2, 1, 0.5, or 0.25 mg/kg) weekly IV for 5 weeks. Physical examination, blood pressure, cardiac output, and electrocardiography (ECG) were evaluated for 4 hours after administration of the first and fifth doses. Blood and urine samples were collected for evaluation of cobalt concentration, CBC and clinical chemistry, and hormone concentrations. Results All mares displayed pawing, nostril flaring, muscle tremors, and straining after CoCl2 infusion. Mares receiving 4, 2, or 1 mg/kg doses developed tachycardia after dosing (HR 60–126 bpm). Ventricular tachycardia was noted for 10 minutes after administration of the 4 mg/kg dose. Increases in systolic arterial pressure (SAP), diastolic arterial pressure (DAP), and mean arterial pressure (MAP) occurred after administration of all doses (4, 2, 1, 0.5, and 0.25 mg/kg). Profound hypertension was observed after the 4 mg/kg dose (SAP/DAP, MAP [mmHg] = 291–300/163–213, 218–279). Hemodynamics normalized by 1–2 hours after administration. ACTH and cortisol concentrations increased within 30 minutes of administration of all CoCl2 doses, and cardiac troponin I concentration increased after administration of the 4 and 2 mg/kg doses. Conclusions and Clinical Importance The degree of hypertension and arrhythmia observed after IV CoCl2 administration raises animal welfare and human safety concerns.

The FGF-23/klotho axis and its relationship with phosphorus, calcium, vitamin D, PTH, aldosterone, severity of disease, and outcome in hospitalised foals

BACKGROUND Fibroblast growth factor-23 (FGF-23) and klotho are key regulators of vitamin D and parathyroid hormone (PTH) synthesis as well as phosphorus and calcium homeostasis; however, information on the FGF-23/klotho axis in healthy and hospitalised foals is lacking. OBJECTIVES The aims of this study were to measure serum FGF-23 and klotho concentrations and determine their association with serum phosphorus, total calcium (TCa), vitamin D metabolite [25(OH)D, 1,25(OH)2 D], PTH, and aldosterone concentrations, disease severity, and mortality in hospitalised foals. STUDY DESIGN Prospective, multicentre, cross-sectional study. METHODS A total of 91 foals ≤72 h old were classified as hospitalised (n = 81; 58 septic; 23 sick non-septic [SNS]) and healthy (n = 10). Blood samples were collected on admission. Hormone concentrations were determined by immunoassays. RESULTS Serum FGF-23, PTH, phosphorus, and aldosterone concentrations were higher while klotho, 25(OH)D, 1,25(OH)2 D, and TCa concentrations were lower in septic and SNS compared to healthy foals (P<0.05). In hospitalised and septic foals, increased FGF-23 and aldosterone concentrations were associated with high phosphorus and PTH but not with TCa and vitamin D metabolite concentrations. Hospitalised foals with the highest FGF-23 and lowest klotho concentrations were more likely to die (odds ratio (OR): 3.3; 95% confidence interval (CI): 1.1-10.3 and OR: 3.1; CI: 1.1-8.0, respectively). MAIN LIMITATIONS Blood gas, ionised calcium, blood culture information not being available for many foals, and use of the sepsis score to classify hospitalised foals. CONCLUSIONS Imbalances in the FGF-23/klotho axis may contribute to mineral dyshomeostasis and disease progression in critically ill foals. Elevated FGF-23 and reduced klotho, together with high phosphorus and PTH concentrations suggests FGF-23 resistance. FGF-23 and klotho are good markers of disease severity and likelihood of mortality in hospitalised foals. Aldosterone may influence phosphorus and PTH dynamics in hospitalised foals. Routine measurement of phosphorus concentrations in sick foals is recommended.